Solution structure of a C-terminal coiled-coil domain from bovine IF(1): the inhibitor protein of F(1) ATPase

Bovine IF(1) is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F(1)F(0) ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF(1) (44-84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.     

The structure of bovine IF(1), the regulatory subunit of mitochondrial F-ATPase.

In mitochondria, the hydrolytic activity of ATP synthase is regulated by an inhibitor protein, IF(1). Its binding to ATP synthase depends on pH, and below neutrality, IF(1) is dimeric and forms a stable complex with the enzyme. At higher pH values, IF(1) forms tetramers and is inactive. In the 2.2 A structure of the bovine IF(1) described here, the four monomers in the asymmetric unit are arranged as a dimer of dimers. Monomers form dimers via an antiparallel alpha-helical coiled coil in the C-terminal region. Dimers are associated into oligomers and form long fibres in the crystal lattice, via coiled-coil interactions in the N-terminal and inhibitory regions (residues 14-47). Therefore, tetramer formation masks the inhibitory region, preventing IF(1) binding to ATP synthase.     

Dimerization of bovine F1-ATPase by binding the inhibitor protein, IF1

In mitochondria, the hydrolytic activity of ATP synthase is regulated by a natural inhibitor protein, IF(1). The binding of IF(1) to ATP synthase depends on pH values, and below neutrality, IF(1) forms a stable complex with the enzyme. Bovine IF(1) has two oligomeric states, dimer and tetramer, depending on pH values. At pH 6.5, where it is active, IF(1) dimerizes by formation of an antiparallel alpha-helical coiled-coil in its C-terminal region. This arrangement places the inhibitory N-terminal regions in opposition, implying that active dimeric IF(1) can bind two F(1) domains simultaneously. Evidence of dimerization of F(1)-ATPase by binding to IF(1) is provided by gel filtration chromatography, analytical ultracentrifugation, and electron microscopy. At present, it is not known whether IF(1) can bring about the dimerization of the F(1)F(0)-ATPase complex.     

Aggregation of wool keratin intermediate filament proteins

The wool keratin intermediate filament proteins were isolated as their S-carboxymethyl derivatives (S-carboxymethylkerateine A, SCMKA) and purified by gel filtration to remove residual non-helical protein of low molecular weight. The alpha-helix content of purified SCMKA was approximately 62% in agreement with that predicted for the alpha-helical coiled-coil segments from the amino acid sequences of the subunits. In aqueous buffer at pH 11 or in n-propanol (20% v/v) at pH 9.2 very large aggregates are dissociated and SCMKA exists largely as a mixture of the dimer (two-chain coiled-coil of Mr approximately 103,000) and the tetramer. The protein species are not in rapidly reversible equilibrium as judged from gel filtration and sedimentation equilibrium. It is probable that species with a range of association constants are present. The equilibrium is shifted towards the dimer with change of pH from 9.2 to 11 or by the addition of 20% (v/v) n-propanol. The tetrameric proteolytic digestion product which is derived from the 1B segment of the alpha-helical rod section of the keratin molecule dissociates in a similar way to intact SCMKA with increase of pH and in the presence of n-propanol. This indicates the importance of this region of the rod domain in the initial stages of the assembly of the filament. Electrostatic and hydrophobic interactions are implicated in the association of the two-chain coiled-coil to the tetramer both in intact SCMKA and the 1B segment tetramer. The results are discussed in relation to the intact dimeric and tetrameric complexes obtained from other intermediate filament types.     

Assembly properties of two CNBr fragments of avian desmin that correspond to the headpiece domain and helix 1B

To study how different domains of the muscle-specific intermediate filament protein, desmin, contribute to its polymerization, two of its CNBr fragments were examined as to their oligomeric structure under assembly conditions. One of these, D88, covers residues 1-88 and represents almost the entire headpiece; the other, D109, covers residues 145-254, and includes the entire Helix 1B and part of linker L12 of the intact molecule. Chemical cross-linking followed by SDS-PAGE, and analytical gel filtration, revealed that in 10 mM Tris-HCl, pH 8.5, conditions that favor tetramerization of intact desmin D88 formed only dimers. D109, on the other hand, formed primarily a dimeric species but low levels of trimeric and tetrameric species were also detectable. These data are consistent with the proposal that, during assembly of intact protein molecules into IF, the headpiece and Helix 1 contribute to dimerization of two polypeptides into a parallel, in-register coiled-coil. However, additional interactions, including headpiece-to-rod binding and hydrophobic interaction along the entire rod domain, are required to stabilize the tetramers and full-size IF.     

Molecular modelling of miraculin: Structural analyses and functional hypotheses

Miraculin is a plant protein that displays the peculiar property of modifying taste by swiching sour into a sweet taste. Its monomer is flavourless at all pH as well as at high concentration; the dimer form elicits its taste-modifying activity at acidic pH; a tetrameric form is also reported as active. Two histidine residues, located in exposed regions, are the main responsible of miraculin activity, as demonstrated by mutagenesis studies. Since structural data of miraculin are not available, we have predicted its three-dimensional structure and simulated both its dimer and tetramer forms by comparative modelling and molecular docking techniques. Finally, molecular dynamics simulations at different pH conditions have indicated that at acidic pH the dimer assumes a widely open conformation, in agreement with the hypotheses coming from other studies.     

Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.     

Deletion of lactose repressor carboxyl-terminal domain affects tetramer formation.

The carboxyl-terminal sequence of the lac repressor protein contains heptad repeats of leucines at positions 342, 349, and 356 that are required for tetramer assembly, as substitution of these leucine residues yields solely dimeric species (Chakerian, A. E., Tesmer, V. M., Manly, S. P., Brackett, J. K., Lynch, M. J., Hoh, J. T., and Matthews, K. S. (1991) J. Biol. Chem. 266, 1371-1374; Alberti, S., Oehler, S., von Wilcken-Bergmann, B., Kr?mer, H., and Müller-Hill, B. (1991) New Biol. 3, 57-62). To further investigate this region, which may form a leucine zipper motif, a family of lac repressor carboxyl-terminal deletion mutants eliminating the last 4, 5, 11, 18, and 32 amino acids (aa) has been constructed. The -4 aa mutant, in which all of the leucines in the presumed leucine zipper are intact, is tetrameric and displays operator and inducer binding properties similar to wild-type repressor. The -5 aa, -11 aa, -18 aa, and -32 aa deletion mutants, depleted of 1, 2, or all 3 of the leucines in the heptad repeats, are all dimeric, as demonstrated by gel filtration chromatography. Circular dichroism spectra and protease digestion studies indicate similar secondary/tertiary structures for the mutant and wild-type proteins. Differences in reaction with a monoclonal antibody specific for a subunit interface are observed for the dimeric versus tetrameric proteins, indicative of exposure of the target epitope as a consequence of deletion. Inducer binding properties of the deletion mutants are similar to wild-type tetrameric repressor at neutral pH. Only small differences in affinity and cooperativity from wild-type are evident at elevated pH; thus, the cooperative unit within the tetramer appears to be the dimer. "Apparent" operator binding affinity for the dimeric proteins is diminished, although minimal change in operator dissociation rate constants was observed. The diminution in apparent operator affinity may therefore derive from either 1) dissociation of the dimeric mutants to monomer generating a linked equilibrium or 2) alterations in intrinsic operator affinity of the dimers; the former explanation is favored. This detailed characterization of the purified mutant proteins confirms that the carboxyl-terminal region is involved in the dimer-dimer interface and demonstrates that cooperativity for inducer binding is contained within the dimer unit of the tetramer structure.     

Inhibitory and Anchoring Domains in the ATPase Inhibitor Protein IF1 of Bovine Heart Mitochondrial ATP Synthase

The inhibitor protein IF1 is a basic protein of 84 residues which inhibits the ATPase activity of the mitochondrial FoF1-ATP synthase complex without having any effect on ATP synthesis. Results of cross-linking and limited proteolysis experiments are presented showing that in the intact FoF1 complex "in situ," in the inner membrane of bovine heart mitochondria, the central segment of IF1 (residues 42-58) binds to the alpha and beta subunits of F1 in a pH dependent process, and inhibits the ATPase activity. The C-terminal region of IF1 binds, simultaneously, to the OSCP subunit of Fo in a pH-independent process. This binding keeps IF1 anchored to the complex, both under inhibitory conditions, at acidic pH, and noninhibitory conditions at alkaline pH.     

The pH-dependent stability of wild-type and mutant transthyretin oligomers

A reduction in pH is known to induce the disassociation of the tetrameric form of transthyretin and favor the formation of amyloid fibers. Using continuum electrostatic techniques, we calculate the titration curves and the stability of dimer and tetramer formation of transthyretin as a function of pH. We find that the tetramer and the dimer become less stable than the monomer as the pH is lowered. The free energy difference is 13.8 kcal/mol for dimer formation and 27 kcal/mol for tetramer formation, from the monomers, when the pH is lowered from 7 to 3.9. Similar behavior is observed for both the wild-type and the mutant protein. Certain residues (namely Glu-72, His-88, His-90, Glu-92, and Tyr-116), play an important role in the binding process, as seen by the considerable pK(1/2) change of these residues upon dimer formation.     

Modeling HIV-1 integrase complexes based on their hydrodynamic properties

We present a model structure of a candidate tetramer for HIV-1 integrase. The model was built in three steps using data from fluorescence anisotropy, structures of the individual integrase domains, cross-linking data, and other biochemical data. First, the structure of the full-length integrase monomer was modeled using the individual domain structures and the hydrodynamic properties of the full-length protein that were recently measured by fluorescence depolarization. We calculated the rotational correlation times for different arrangements of three integrase domains, revealing that only structures with close proximity among the domains satisfied the experimental data. The orientations of the domains were constrained by iterative tests against the data on cross-linking and footprinting in integrase-DNA complexes. Second, the structure of an integrase dimer was obtained by joining the model monomers in accordance with the available dimeric crystal structures of the catalytic core. The hydrodynamic properties of the dimer were in agreement with the experimental values. Third, the active sites of the two model dimers were placed in agreement with the spacing between the sites of integration on target DNA as well as the integrase-DNA cross-linking data, resulting in twofold symmetry of a tetrameric complex. The model is consistent with the experimental data indicating that the F185K substitution, which is found in the model at a tetramerization interface, selectively disrupts correct complex formation in vitro and HIV replication in vivo. Our model of the integrase tetramer bound to DNA may help to design anti-integrase inhibitors.     

Kinetic study on the dimer-tetramer interconversion of glycogen phosphorylase a.

Kinetic theory of dissociating enzyme systems has been applied to a study of the dimer-tetramer interconversion of glycogen phosphorylase a. All kinetic constants for the dissociating-associating reaction of phosphorylase a have been determined. The results indicate that (a) the presence of glucose-1-phosphate has no influence on either the rate of dissociation or the rate of association, and hence does not shift the dimer-tetramer equilibrium of phosphorylase a; (b) the binding og glycogen to the enzyme decreases the association rate of the dimer to form the tetramer, but has no effect on the dissociation rate of the tetramer; (c) both the dimeric and tetrameric form of phosphorylase a can bind glycogen, but the tetrameric form has a lower affinity for glycogen and is catalytically inactive.     

Structural aspects of aldehyde dehydrogenase that influence dimer-tetramer formation

Aldehyde dehydrogenases are isolated as dimers or tetramers but have essentially identical structures. The homotetramer (ALDH1 or ALDH2) is a dimer of dimers (A-B + C-D). In the tetrameric enzyme, Ser500 from subunit "D" interacts with Arg84, a conserved residue, from subunit "A". In the dimeric ALDH3 form, the interaction cannot exist. It has been proposed that the formation of the tetramer is prevented by the presence of a C-terminal tail in ALDH3 that is not present in ALDH1 or 2. To understand the forces that maintain the tetramer, deletion of the tail in ALDH3, addition of different tails in ALDH1, and mutations of different residues located in the dimer-dimer interface were made. Gel filtration of the recombinantly expressed enzymes demonstrated that no change in their oligomerization occurred. Urea denaturation showed a diminution to the stability of the ALDH1 mutants. The K(m) for propionaldehyde was similar to that of the wild-type enzyme, but the K(m) for NAD was altered. A double mutant of D80G and S82A produced an enzyme with the ability to form dimers and tetramers in a protein-concentration-dependent manner. Though stable, this dimeric form was inactive. The tetramer exhibited 10% activity compared with the wild type. Sequence alignment demonstrated that the hydrophobic surface area is increased in the tetrameric enzymes. The hydrophobic surface seems to be the main force that drives the formation of tetramers. The results indicated that residues 80 and 82 are involved in maintaining the tetramer after its assembly but the C-terminal extension contributes to the overall stability of the assembled protein.     

Quaternary structure of the Mr 46,000 mannose 6-phosphate specific receptor: effect of ligand, pH, and receptor concentration on the equilibrium between dimeric and tetrameric receptor forms

The Mr 46,000 mannose 6-phosphate specific receptor exists in solution as a mixture of noncovalently associated dimeric and tetrameric forms. The two quaternary forms were separated by sucrose density centrifugation, and their composition was assessed by cross-linking with bifunctional reagents followed by SDS-polyacrylamide gel electrophoresis. The dependence of equilibrium between the dimeric and tetrameric forms on pH, receptor concentration, and presence of mannose 6-phosphate was studied. The formation of tetrameric forms is favored by pH values around 7, high receptor concentration, and presence of mannose 6-phosphate ligand. Tetrameric forms bind stronger at pH 7 to phosphomannan-Sepharose 4B than dimeric forms. Both quaternary forms dissociate at the same pH from a mannose 6-phosphate affinity matrix. When starting with dimeric or tetrameric forms, the equilibrium between dimeric and tetrameric forms is reached at pH 7.5 and 4 degrees C after 6-8 days. The presence of 5 mM mannose 6-phosphate shifts the equilibrium toward tetrameric forms. At pH 4.5 and 4 degrees C, the association of dimeric to tetrameric forms is negligible, while tetrameric forms dissociate to dimeric forms within 12 h. The results demonstrate that oligomerization is an intrinsic property of MPR-46 that is affected by ligand binding, pH, and receptor concentration.     

Fructose-1,6-bisphosphatase from Synechococcus leopoliensis. Substrate-dependent dimer-tetramer interconversion

Extracts of Synechococcus leopoliensis (Anacystis nidulans) contain two forms of D-fructose-1,6-bisphosphatase (EC 3.1.3.11) previously designated as forms A and B [Gerbling, K.-P., Steup, M., and Latzko, E. (1984) Arch. Microbiol. 137, 109-114]. Form B, which probably represents the major part of the total extractable fructose-1,6-bisphosphatase activity, has been purified to apparent homogeneity. Gel filtration, non-denaturing polyacrylamide gel electrophoresis, and cross-linking with bis(sulfosuccinimidyl)suberate revealed that the fructose-1,6-bisphosphatase B exists in either a dimeric or in a tetrameric subform, depending upon the absence or presence of fructose-1,6-bisphosphate and Mg2+. The dimer--tetramer interconversion was readily reversible. The results provide evidence for a two-step activation of fructose-1,6-bisphosphatase B involving the reduction of the dimeric subform and the subsequent substrate-dependent conversion of the reduced dimer to a reduced tetramer, which is the only catalytically active state. In contrast to form B, no substrate-dependent interconversion was detected with form A from S. leopoliensis.     

NADP(+)-malic enzyme from sugarcane leaves: structural properties studied by thermal inactivation

The irreversible thermal inactivation of the sugarcane leaf NADP(+)-malic enzyme was studied at 50 degrees C and pH 7.0 and 8.0. Depending on the preincubation conditions, thermal inactivation followed mono- or biphasic first-order kinetics. A two-step behavior in the irreversible denaturation process was found when protein concentration was sufficiently low. The protein concentration necessary to obtain monlphasic thermal inactivation kinetics was lower at pH 8.0 than at pH 7.0. The results suggest that biphasic inactivation kinetics are the consequence of the existence of two different oligomeric forms of the enzyme (dimer and tetramer), with the dimer being more stable in regards to thermal inactivation. The effects of the substrate and essential cofactors on the thermostability and equilibrium between the dimeric and tetrameric enzyme forms were also studied. Depending on the pH, NADP+, L-malate, and Mg2+ all had a protective effect on the stability of the dimeric and tetrameric species during thermal treatment. However, these ligands showed different effects on the aggregation state of the enzyme. NADP+ and L-malate induced dissociation, especially at pH 8.0, whereas Mg2+ induced aggregation of the protein. By studying the thermal inactivation kinetics at 50 degrees C and different pH values it was observed that the equilibrium between dimers and tetramers was dramatically affected in the range of pH 7.0-8.0. These results suggest that an amino acid residue(s) in the protein with an apparent pKa value of 7.7 needs to be deprotonated to stabilize aggregation of the enzyme to the tetrameric form.     

Glutamic acid in the inhibitory site of mitochondrial ATPase inhibitor, IF(1), participates in pH sensing in both mammals and yeast

The mitochondrial ATPase inhibitor, IF(1), regulates the activity of F(1)F(o)-ATPase. The inhibitory activity of IF(1) is highly pH-dependent. The effective inhibition by IF(1) requires a low pH. Under basic conditions, its activity markedly declines. The importance of His49 in the pH dependence of bovine IF(1) is well-known. However, the residue is not conserved in yeast IF(1). We previously showed that Glu21 is required for the pH dependence of yeast IF(1), but the function of homologous Glu in mammalian IF(1) is not clear. In this study, we examined the requirement for Glu26 of bovine IF(1) (corresponding to Glu21 of yeast IF(1)) regarding its pH dependence by amino acid replacement. Three mutant proteins, E26A, H49K and the double mutant E26A/H49K, were overexpressed and purified. All mutants retained their inhibitory activity well at pH 8.2, although wild-type IF(1) was approximately 10-fold less active at pH 8.2 than at 6.5. A covalent cross-linking study revealed that both wild-type IF(1) and the E26A mutant formed a tetramer at pH 8.2, although H49K and E26A/H49K mutants did not. These results indicate that, in addition to His49, Glu26 participates in pH sensing in bovine IF(1), and the mechanism of pH sensing mediated by Glu26 is different from the dimer-tetramer model proposed previously.     

Dimeric interaction between the cytoplasmic domains of the Na+/H+ exchanger NHE1 revealed by symmetrical intermolecular cross-linking and selective co-immunoprecipitation

To investigate the oligomeric structure of Na(+)/H(+) exchanger 1 (NHE1), permeabilized cells and membranes from cells expressing NHE1 variants were treated with the oxidizing agent Cu(2+)/o-phenanthroline or the bifunctional sulfhydryl reagent methanethiosulfonate. These treatments resulted in symmetrical intermolecular cross-linking at intrinsic (Cys(794) and Cys(561)) or 15 exogenous cysteine residues introduced into the distal carboxyl- (C-) terminal cytoplasmic domain (after aa 600) but not at intrinsic Cys(538) because of masking by its tight association with calcineurin B-homologous protein. Cross-linking was abolished in membranes solubilized with sodium dodecyl sulfate, which dissociates oligomeric NHE1, while it was preserved in those treated with Triton X-100. In addition, treatment with cross-linkers did not produce the tetrameric forms of NHE1 mutants with two cysteine residues. Thus, cross-linking presumably occurs between adjacent C-termini of the NHE1 dimer but not by a stochastic process via random collision of NHE1 molecules. The observations suggest that at least the distal C-termini of the NHE1 dimer are flexible or mobile and are thereby capable of easily making contact with each other over the large cytoplasmic portion of the molecule. Furthermore, co-immunoprecipitation experiments showed that the proximal C-termini (aa 503-580) have a strong propensity to interact directly with each other in parallel. Deletion of aa 562-579 resulted in disruption of disulfide cross-linking between the C-termini and markedly reduced the intracellular pH sensitivity of Na(+)/H(+) exchange, suggesting that the dimeric interaction in this region may control the pH-dependent regulation of NHE1.     

The molecular mechanism for the tetrameric association of glycogen phosphorylase promoted by protein phosphorylation.

The allosteric transition of glycogen phosphorylase promoted by protein phosphorylation is accompanied by the association of a pair of functional dimers to form a tetramer. The conformational changes within the dimer that lead to the creation of a protein recognition surface have been analyzed from a comparison of the crystal structures of T-state dimeric phosphorylase b and R-state tetrameric phosphorylase a. Regions of the structure that participate in the tetramer interface are situated within structural subdomains. These include the glycogen storage subdomain, the C-terminal subdomain and the tower helix. The subdomains undergo concerted conformational transitions on conversion from the T to the R state (overall r.m.s. shifts between 1 and 1.7 A) and, together with the quaternary conformational change within the functional dimer, create the tetramer interface. The glycogen storage subdomain and the C-terminal subdomain are distinct from those regions that contribute to the dimer interface, but shifts in the subdomains are correlated with the allosteric transitions that are mediated by the dimer interface. The structural properties of the tetramer interface are atypical of an oligomeric protein interface and are more similar to protein recognition surfaces observed in protease inhibitors and antibody-protein antigen complexes. There is a preponderance of polar and charged residues at the tetramer interface and a high number of H-bonds per surface area (one H-bond per 130 A2). In addition, the surface area made inaccessible at the interface is relatively small (1,142 A2 per subunit on dimer to tetramer association compared with 2,217 A2 per subunit on monomer-to-dimer association).     

Regulation of phosphoenolpyruvate carboxylase from Crassula by interconversion of oligomeric forms

Using size-exclusion high-performance liquid chromatography, it is shown that phosphoenolpyruvate carboxylase from Crassula argentea, a crassulacean acid metabolism (CAM) plant, exists primarily in the form of a tetramer of a 100-kDa subunit at night and as a dimer of the same subunit during the day. The tetrameric enzyme from night leaves is not inhibited by malate, while the dimeric form from day leaves can be completely inhibited by malate. The purified day, or dimer, form of the enzyme can be converted to the tetramer by concentration and exposure to Mg2+. When thus converted, the tetramer is insensitive to malate inhibition, and is more strongly activated by glucose 6-phosphate than the dimer. The purified night, or tetramer, form is converted to the dimer by incubation for 60 min at pH 8.2. This enzyme may also be converted to the dimer by adding 1.5 mM malate to the elution buffer, but preincubation for 15 min with phosphoenolpyruvate prevents disaggregation when chromatographed with buffer containing malate. Preincubation with 1mM EDTA and subsequent chromatography with buffer containing malate shows a progressive dissociation of the tetrameric form with increasing time of preincubation. The implications of these observations for the diurnal regulation of phosphoenolpyruvate carboxylase in CAM metabolism are discussed.